1. Field of the Invention
The invention relates generally to a subsystem for fluorescence and absorption spectroscopy and more specifically to an apparatus for simultaneously generating absorption and fluorescent data for a single sample.
2. Description of the Prior Art
High performance liquid chromatography (HPLC), gel electrophoresis and capillary electrophoresis are all used for biochemistry applications such as separation of DNA molecules. Electrophoresis is a separation technique produced by the migration of charged molecules (or particles) in an electrolyte under the influence of an electric field. Smaller or more highly charged sample molecules move faster than larger or lower charged molecules. Hence, each species of the sample molecules is divided into bands which pass or reach a fixed point at different times.
In gel electrophoresis (GE), the electrolyte is usually supported by a porous hydrophilic polymer matrix, the gel, coated on a sheet of glass, sandwiched together with another glass plate and sealed on each side with a gasket. The samples are applied to the top edge of the gel. The bottom edge of the gel sandwich is placed vertically in a reservoir containing a buffered electrolyte. A second reservoir is placed on top of the sandwich and filled with buffered electrolyte. Each reservoir contains an electrode connected to the proper output of a DC power supply. A voltage of up to two thousand volts is applied to the sample. A typical GE run may take six hours. After the run the plate must be stained to visualize the GE bands of interest.
The success of electrophoresis in most applications depends upon the effective utilization of a stabilizing medium such as polymer gels. The gels stabilize the separation medium against convection and flow which would otherwise disrupt separations. A large body of modern electrophoresis technology is devoted to understanding and to controlling the formation of electrophoresis gels.
Gel electrophoresis, as commonly practiced, is generally not considered a true instrumental method of analysis. Instrumental versions of gel electrophoresis analogous to column chromatography are still in the developmental stages in most cases. The presence of the stabilizing gels has prevented the adaptation of electrophoresis to on-line detection quantification or automated operation and consequently gel electrophoresis is still a manual intensive methodology.
Capillary electrophoresis has been developed as an alternative to column chromatography and gel electrophoresis because capillary electrophoresis is up to ten times faster than gel electrophoresis and is more accurate. In capillary electrophoresis, the time, expense and variability of packing a chromatographic column or casting an electrophoresis gel are obviated. Small samples are separated and analyzed in a few minutes. The sharpness of separations is enhanced by the use of narrow-bore tubing, since this minimizes the thermal gradients and the consequential convective turbulence and diffusion of sample components. Capillary separations avoid the "eddy migration" problems which are encountered when stabilizing media such as electrophoretic gels or chromatographic packings are used.
A schematic of a prior art capillary electrophoresis system is illustrated in FIG. 1. A high voltage power supply 10 provides a high voltage to a first electrode 17 which is mounted in a first reservoir 16 containing an electrolyte. A first end 15 of a capillary tube 14 is also suspended in reservoir 16. A second end 13 of capillary tube 14 is mounted in a second reservoir 12 containing an electrolyte and a second electrode 11 connected to ground through power supply 10. A detector 18 is mounted around capillary tube 14.
Capillary tube 14, used to bridge the gap between two electrolyte reservoirs 12, 16, is typically a fused silica capillary tube 50 microns in diameter and about 50 cm long. Tube 14 is first filled with electrolyte and then approximately five nanoliters of sample solution are introduced at end 15 of capillary tube 14, and an ultraviolet (UV) light is passed through the diameter of the capillary tube by the detector near end 13 of tube 14. When a 20-30 KV potential is applied across capillary tube 14, electrophoresis causes all charged sample molecules to travel along capillary tube 14 at different velocities and to pass through the illuminated section of capillary tube 14 at different times. The sample molecules are detected by a photosensor placed opposite the UV light source in the case of an absorbance detector, or at right angles to the light source in the case of a fluorescence detector. All sample components whether anions, cations, or neutrals are eventually swept through the detector as peaks, sometimes called bands, and the output signals from the detector are analyzed to identify the characteristics of the sample.
Since the migration time for a species in the sample is dependent upon the length of capillary tube 14, the electrophoretic mobility of the species, and the applied voltage, species having different electrophoretic mobilities will pass through the detector at different times. The separation efficiency of the various species in the sample in terms of the total number of theoretical plates is dependent upon the mobility, the applied voltage and the diffusion coefficient. Hence, high separation efficiencies are best achieved through the use of high voltages. Also, column length plays no role in the separation efficiency but column length has an influence on the migration times and hence the time required for analysis of a sample.
The CE system, as illustrated in FIG. 1, potentially provides rapid, high resolution online detection capability that is not attainable wih gel electrophoresis equipment. Capillary electrophoresis circumvents the labor intensive manual procedures of experiment preparation, sample manipulation, data generation and interpretation which is inherent in gel or other stabilizing medium electrophoresis techniques. Further, the quantity of sample required to perform capillary electrophoresis is significantly less than that of the other methods.
A variety of methods for introducing the sample into the capillary tube have been used. Displacement techniques such as direct injection, gravity flow or siphoning and suction are commonly used since these techniques are simple and produce separations which accurately reflect the relative concentration of sample constituents. Other techniques for introducing the sample involve the principle of electromigration. In these applications, the samples are introduced into the capillary tube by a short duration electrical current. Both electrophoretic and electroendosmotic forces can contribute to the sample movement in these techniques. Devices using a sample splitter (See M. Deml, F. Foret and P. Bocek, "Electric Sample Splitter for Capillary Zone Electrophoresis," J. Chrom. 346 pp. 159-165 (1985)), a micro injector (See R. A. Wallingford and A. G. Ewing, "Characterization of a Micro Injector for Capillary Electrophoresis," Anal. Chem. 59, pp. 678-681 (1987)), and a rotary injector (See T. Tsuda, T. Mizuno, and J. Akiyama, "Rotary-Type Injector for Capillary Zone Electrophoresis," Anal. Chem. 59, pp. 799-800 (1987)) have been reported.
In capillary electrophoresis separations utilizing electroendosmotic flow, the sample components are introduced at the high voltage anode side of the apparatus. This is the opposite of most conventional electrophoretic techniques.
Fused silica is most commonly used for electrophoretic capillaries. Capillary tube inner diameters of 50-100 microns with wall thicknesses of less than 200 microns are used in most applications. Capillary lengths of 10-100 centimeters are most often used. While as described above, the species separation is theoretically independent of capillary length, and a shorter tube would seem advantageous in minimizing band broadening caused by diffusion and sample interactions. Practical considerations of Joule heat dissipation dictate the length of the capillary tube.
The regulated direct current high voltage power supplies have had potentials up to 50 kilovolts. These voltages generate microamp currents through the capillary tube. Again, while the theoretical considerations indicate that faster separations are obtained with higher voltage potentials, there are practical limits imposed by heat dissipation requirements. In addition, excessively high voltages may result in corona discharge through the capillary tube and elsewhere within the instrument.
The small scale of analysis in capillary electrophoresis requires ultra-sensitive detection instrumentation. Hence, no convenient universal detector for every conceivable type of sample molecule exists. Multiple detection methodologies have been used. Capillaries have been used for years in "on-column" detection among several areas of the separation sciences, most notably, liquid and gas chromatography. Hence, much of the detection instrumentation for capillary electrophoresis is drawn from these areas. Ultraviolet wave length detectors are commonly used for analysis of amino acids, peptides, proteins, nucleics components, as well as some carbohydrates, drugs and other molecules of biological significance. In one prior art application, capillary electrophoresis zone detection was accomplished with fluorescence detectors and ultraviolet absorption detectors. Both detectors were separately used in an on-column mode (See J. W. Jorgenson and K. D. Lukacs, "Capillary Zone Electrophoresis," Science, Vol. 222, pp. 266-272, Oct. 21, 1983).
Commercial absorption detectors and fluorescence detectors are available which may be modified for use in capillary electrophoresis. In one prior art absorption detector, shown in FIG. 2, a low wavelength ultraviolet light 20, typically between 200 nanometers and 280 nanometers, is incident upon a diffraction grating 21. Grating 21 separates the light into different wavelength components and one of these components 24 is passed through the sample measurement region 23 of capillary tube 22. The light 25 emerging from the sample is incident upon a first photodiode 26. Actually, as shown in FIG. 2, the light from grating 24 is divided into two parts, one which passes through the sample and a second which is incident upon a second photodiode 27. The signal from photodiode 27 is used as a reference so that as the relative intensity from light source 20 changes, the signal from first photodiode 26 is corrected for the changes in light source 20.
This apparatus has two primary limitations. First, only one wavelength at a time is incident upon the sample. To use a second wavelength, grating 21 must be mechanically repositioned so that a different wavelength is incident upon capillary tube 22. The second problem with this absorption detector is designing a holder for capillary tube 22 so that capillary tube 22 is not damaged while performing the measurement, changing samples, or loading the sample into the capillary tube.
In prior art fluorescence detectors, an ultraviolet (UV) light source, having a selected wavelength, illuminates the sample. The wavelength of the ultraviolet light causes certain molecules to fluoresce and emit light at wavelengths different from the wavelength of the incident UV light. A spectrophotometer is oriented at an angle, typically 90.degree., from the light source so that the emitted visible light (between 450 and 630 nm) is measured against a black background.
Fluorescence detection is inherently very sensitive and biomolecules can be detected using fluorescence via a fluorescence tag for proteins or a "stain" for DNA. The spectrophotometer, used in fluorescence detectors, typically has a grating and a photomultiplier tube. The grating reflects a specific wavelength of the fluorescent light from the sample upon a photomultiplier tube. The photomultiplier tube provides great sensitivity, but using such a tube inherently restricts the detector to a single channel. Rotation of the grating is required for the selection of a different wavelength.
To overcome the limitations of single wavelength operation, a detector has been developed which utilizes a spectrophotometer and a diode array. In this detector, the light from the sample is incident upon a grating. The grating disperses the light from the sample into a spectrum of wavelengths. Instead of having a single photodiode or photomultiplier tube to intercept the light from the grating, an array of up to 1000 photodiodes on a single semiconductor chip is used.
In this self scanning diode array detector, electronic sensing circuitry measures the charge on a capacitor associated with each diode by quickly scanning the capacitors with a video type signal. Thus, the relative light intensity incident upon each diode is measured. Each diode in the array corresponds to two nanometers in bandwidth and so the measurement provides a complete spectrum. However, the rapid switching from one diode to another introduces electronic noise, which in turn limits the sensitivity of the detector. Thus, this detector generates a spectrum from which information about the chemistry of the sample can be ascertained by looking at the relative absorption at different wavelengths, but the detector does not provide the sensitivity of single channel detectors.
The prior art detectors are not easily adapted for use in capillary electrophoresis. Each detector requires a special holder for the capillary tube and the combination of the thin capillary tube and the requirement for changing electrolytes and samples makes an automated apparatus impractical. While as previously described, capillary electrophoresis has significant advantages over gel electrophoresis, the detector limitations inhibit the development of an automated instrument.
A detector which provides the sensitivity of the single channel absorption detector or fluorescence detector and the advantages of a multichannel detector is currently not available. Further, separate detectors are used for fluorescence and absorption measurements. Thus multiple tests are required to obtain fluorescence and absorption data. A system having an integral absorption and fluorescence detector with multiple channel capability would significantly enhance the flexibility and utilization of capillary electrophoresis and in fact all HPLC.
While capillary electrophoresis is faster and more accurate than gel electrophoresis, prior art systems still require multiple manual manipulations. For example, manual filling and replacement of the electrolyte is frequently required during electrophoresis tests. Accordingly, capillary electrophoresis measurements, while not as manually intensive as the gel electrophoresis measurements, still require some amount of manual intervention.